1. Field of the Invention
The present invention relates to a novel antibody or antibody fragment comprising a cysteine residue (hereinafter referred to as Cys residue), to which a hydrophilic macromolecular group or amphipathic macromolecular group can be bound at a high efficiency. In addition, the present invention also relates to a novel antibody modified product or antibody fragment modified product in which the Cys residue is chemically modified.
2. Brief Description of the Background Art
Since antibodies have high binding affinity, binding specificity and high stability in blood, application of the antibodies as diagnostic agents or therapeutic agents for human are progressed (Non-patent Reference 1). As the background of advance in the application of therapeutic antibodies, preparation of human chimeric antibodies and humanized antibodies making use of genetic engineering is considered (Non-patent References 2 to 5). A human chimeric antibody comprises an antibody variable region derived from an antibody of an animal other than human, and a constant region derived from a human antibody.
A humanized antibody comprises complementarity-determining regions (CDR) derived from an antibody of an animal other than human in variable region of the antibody, and the remainder of framework regions (FR) and the constant region derived from a human antibody. Due to this, problems relating to the non-human animal antibodies such as high immunogenicity and low effector activity would be solved.
However, since such antibodies are giant molecules having a molecular weight of exceeding 100,000, their transition from blood into tissues is considerably slow. Accordingly, studies are carrying out on an antibody fragment having an increased transitional activity in the living body and a lower molecular weight. The high specificity and affinity in an antibody therapy can be depending on CDRs of antibody variable region.
As the antibody fragment having an antibody variable region, for example, various shapes such as Fv, Fab, Fab′, F(ab′)2, single chain antibody (scFv), dimerized V region (diabody), and disulfide stabilized V region (dsFv) are known, but the short blood half life accompanied by lowering of the molecular weight comes to be a serious problem.
As a method for solving this problem, there is an antibody fragment modified product modified by a hydrophilic macromolecule group or amphipathic macromolecule group such as polyethylene glycol (PEG). It is possible to adjust a blood half life from several minutes to several hours by increasing the average molecular weight of PEG, and in the case of Fab, it became possible to obtain a blood half life equivalent to the corresponding antibody by binding Fab to PEG having a molecular weight of 40 kDa (Non-patent Reference 6).
The exiting method for modifying Fab fragment with PEG is a method in which a Cys residue contributing to a disulfide bond at the C-terminal site is used as a binding region or a method in which a Cys residue contributing to a disulfide bond at the hinge region is used as a binding region by further elongating the C-terminal site of Fab fragment by the hinge region. However, in each case of the fragments, it is difficult to obtain the Cys residue under free form in the expression and purification steps. Thus, a reduction step is necessary as a pretreatment of the PEG modification (Non-patent Reference 6).
On the other hand, an antibody-drug conjugate (ADC) has been drawing attention as a new antibody derivative making use of the high binding specificity of antibody (Non-patent References 7 and 8). ADC is possible to deliver a drug as one of the functional molecules loaded on the antibody derivative, specifically into a target cell by endocytosis of a target antigen upon binding the antibody.
Although effector function of the antibody has a mechanism of action outside of a cell mediated by an immune system, since ADC has an intracellular mechanism of action, it is possible to use it depending on biological characteristics of a target antigen. For example, in the United States, Mylotarg (registered trademark) (Gemtuzumab Ozogamicin) has been approved as an ADC for the first time in the world. In addition, in the Phase II trial of Tratsuzumab-DM1 on Her2-positive progressive breast cancer patients, reduction of the cancer has been found in 25% of the patients. Accordingly, progress in the developing state of ADC is remarkable, and it is expected to be a new form of pharmaceuticals in the future.
According to a result of study using a cell line, drug efficacy of ADC is related to both the potency of the drug and the number of bond of the loaded drugs. However, in the case of a hydrophobic drug, a problem of considerably lowering its stability in blood has been found in a drug efficacy test using animal individual, due to increase in drug-dependent hydrophobic property (Non-patent Reference 9).
Since PEG has high hydration property, it is possible to improve hydrophobic property by adjusting the molecular weight depending on the drug to be used. As a method for solving this problem, development of a drug comprising a hydrophilic macromolecule or amphipathic macromolecule such as PEG has also been started, it is expected that the development of a hydrophilic or amphipathic molecule for the purpose of enhancement of drug efficacy of ADC will be progressed greatly (Patent References 1 and 2)).
In the existing ADC, a drug is covalently bound to an α-amino group of the N-terminal, an ε-amino group of a lysine (Lys) residue or a thiol group of a Cys residue in the antibody molecule or antibody fragment molecule. However, when two or more drugs are introduced into one antibody molecule, since generally it is necessary to bind them to amino acid residues having different reactivity, a heterogeneous mixture having different numbers of drugs is formed depending on the reaction conditions such as the reaction scale, the number of equivalences and the like. Thus, it also accompanies a difficulty in constructing a production process.
The thiol group comprised in the Cys residue among natural amino acids is an ideal functional group in order to carry out the reaction under mild conditions because it has high reactivity even at a neutral pH range. In general, since it shows its higher reactivity for an electrophilic reaction reagent than that of α-amino group of the amino-terminal or the ε-amino group of a Lys residue inside the protein, or the hydroxyl group derived from a serine (Ser) residue or a threonine (Thr) residue, it is possible to control the reaction site easily.
Accordingly, when an antibody or antibody fragment is chemically modified with a hydrophilic macromolecule or amphipathic macromolecule such as PEG or with a functional molecule such as a drug, by introducing one or more of a stable free Cys residue into a specified site of the antibody or antibody fragment, particularly into the constant region, further efficient chemical modification, reduction of the number of steps and avoidance of structural instabilization accompanied by the disulfide bond destruction due to the reduction operation can be expected.
Conventionally, when a protein is expressed in the periplasmic space of Escherichia coli or in the culture supernatant of a eukaryotic cell, since the protein-derived Cys residue or the Cys residue introduced by artificially substituting an amino acid residue is affected by the formation of intermolecular disulfide bond, S-glutathione formation and the like, it was difficult to be substituted with the free Cys residue having reactivity (Non-patent References 10 and 11).
In addition, regarding the substitution for a Cys residue in an antibody molecule, it has been reported that the ratio of the free Cys residue of inside the antibody molecule is higher than that of the surface of the antibody molecule, but the ratio of free Cys residue to all of the substituted Cys residues is approximately 50% at the maximum (Non-patent Reference 12 and Patent Reference 3).
On the other hand, it has also been reported a method for substituting a free Cys residue for a structural region having a high ratio of solvent accessible surface area or a residue having a structure close to the Cys residue, such as a Ser residue or a Thr residue, based on the structural information (Patent References 4 to 6), but it is necessary to carry out a reduction treatment in order to obtain a free Cys residue.
In addition, a substitution site to a free Cys residue in which a low molecular maleimide-biotin complex is modified at a high efficiency of 60 to 100%, by the PHESELECTOR assay, using a phage system (Non-patent Reference 13 and Patent Reference 7) has been found, but there is no description on the modification efficiency with a hydrophilic macromolecular group or amphipathic macromolecular group such as PEG.